肝癌异常表达microRNAs的鉴定、功能、作用靶点以及治疗潜力研究
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摘要
目的原发性肝细胞癌(hepatocellular carcinoma; HCC;肝癌)是一种全球性人类恶性肿瘤,具有高发病率和高死亡率特点,但缺乏有效的靶向治疗。当前诊断肝癌的生物标志物主要是甲胎蛋白(AFP),但有20%到40%的患者AFP呈阴性或低度阳性。手术切除或肝移植仍然是目前治疗肝癌的首选,但只有不到20%的肝癌病人适合外科治疗,并且术后复发率高,5年存活率只有30%到40%。肝癌病人的结局如此凄凉的主要原因是我们对肝癌发病的分子机制仍知之甚少和缺乏有效的靶向治疗。因此,致力于阐明肝癌的发生发展机制,探索新的早期诊断生物标志物和更有效的治疗方法是一项十分紧迫的研究课题。MicroRNAs (miRNAs)是近年新起的一个研究领域,为一组保守的非编码小RNA,通过转录后水平调控基因的表达。MiRNAs表达水平的改变能够调节肿瘤发生相关的重要生物学过程,因而系统评价某种确定表型肿瘤miRNA表达谱的变化将有利于我们更深入的理解肿瘤发生发展的机制。本研究拟应用肝癌细胞细胞株与原代正常肝细胞miRNA表达谱差异这一全新的策略鉴定出肝癌异常表达的miRNAs,并进一步系统的研究这些miRNAs的功能、作用靶点以及治疗潜力。
方法应用Agilent human miRNA array分析7株肝癌细胞株和3例原代正常肝细胞的miRNA表达谱,通过比较得到肝癌中异常表达的miRNAs,并用TaqMan qRT-PCR进行确认。在细胞水平上转染miRNA前体或抑制剂以及相应的对照miRNA,通过细胞增殖、细胞周期和凋亡分析、细胞迁移与侵袭以及克隆形成实验,探讨这些异常表达的miRNAs在肝癌发生发展中的作用。利用生物信息学技术如miRBase、TargetScan、PicTar等数据库,预测miRNA的作用靶点,再通过SYBR Green定量PCR、Western Blot以及Luciferase精确靶点实验,验证肝癌相关miRNAs的作用靶点,从而阐明这些异常表达的miRNAs在肝癌发生中,类似于抑癌基因或癌基因的分子作用机制。利用肝癌裸鼠移植瘤模型,探索这些异常表达的miRNAs分子或其拮抗剂在肝癌治疗中的潜力。
结果通过比较肝癌细胞株与原代正常肝细胞的miRNA表达谱差异获得肝癌异常表达miRNAs 37个(2-fold change,P< 0.05)。我们证实上调的miR-221和下调的miR-375通过调控细胞增殖、周期、凋亡、迁移侵袭以及克隆形成参与了肝癌的发生发展。miR-221作用其靶点BMF、BBC3和ANGPTL2而作用类似于癌基因,miR-375作用其靶点LAMC1和PDGFC而作用类似于抑癌基因。治疗性的应用慢病毒介导的anti-miR-221或胆固醇修饰的miR-375类似物可以有效的抑制肝癌裸鼠移植瘤的生长。
结论通过比较肝癌细胞株与原代正常肝细胞的miRNA表达谱差异可以高效鉴定出肝癌特异性的miRNAs。这些异常表达的miRNAs通过作用其靶点,调节了肿瘤发生相关的重要生物学过程,从而参与了肝癌的发生和发展。实验结果显示慢病毒介导的anti-miR-221或胆固醇修饰的miR-375类似物对治疗肝癌有良好的潜力。所有这些,使我们相信基于miRNAs的诊断和治疗在抗肝癌研究中具有诱人的前景。
Objective Hepatocellular carcinoma (HCC) is a worldwide human malignancy with high morbidity and mortality but lack of effective targeted therapies. Current diagnosis biomarkers of HCC are maily depend on alpha-fetoportien (AFP), but 20%to 40%HCC patients'AFP was negative or low positive. Up to now, surgical resection or liver transplantation remains the first choice for the treatment of HCC; however, only about 20% of patients with HCC are eligible for surgical intervention at the time of diagnosis. Moreover, patients who underwent curative resection often have a high frequency of relapse, and postoperative 5-year survival is only 30%-40%. The outcomes of HCC patients are still so dismal due to our limited knowledge in its molecular pathogenesis, the difficulty in detecting the disease at its early stages and the lack of effective therapeutics. Therefore, studies aimed at the definition of the mechanisms associated with hepatocarcinogenesis, identification of new biomarkers for early diagnosis and development of more effective therapeutic interventions are urgently needed. MiRNAs are evolutionarily conserved, small noncoding RNAs that are believed to play fundamental roles in various biological processes through regulation of gene expression at the level of post-transcription. Alterations in miRNA expression can modulate key cellular processes involved in tumorigenesis; so systematic evaluation of changes in global miRNA expression in phenotypically defined tumors could provide insight into basic mechanisms of tumor formation and progression. In this report, we systematically studied the aberrantly expressed miRNAs involved in HCC through the comparison of miRNA expression profiling in cancerous hepatocytes with that in normal primary human hepatocytes and emphasized the functions, targets and therapeutic potential of these aberrantly expressed miRNAs.
Methods The expression profiles containing 723 human miRNAs were determined in 7 liver cancer cell lines and 3 cases of normal primary human hepatocytes (PHHC) using Agilent human miRNA array (Version 2). MiRNA expression profiles of cancerous hepatocytes were compared with that of normal hepatocytes to indentify the aberrantly expressed miRNAs. These results were further confirmed by TaqMan qRT-PCR. To assess the role of dysregulated miRNAs in hepatocarcinogenesis, hepatoma cell lines were transfected with pre-miRNAs, anti-miRNAs or their matched negative control and further tested by cell proliferation, cell cycle and apoptosis, clonogenicity, cell migration and invasion assaies. The putative miRNA targets were predicted by using algorithms miRBase, TargetScan and PicTar and verfied by SYBR Green qRT-PCR, Western Blot Analysis and Luciferase Assay. The model of hepatoma xenografts in nude mice was established to explore the therapeutic potential of antisense-mediated inhibition of oncogenic miRNAs or replacement therapy of tumor suppressor miRNAs for HCC.
Results we reported a previously unrecognized paradigm for identifying aberrantly expressed miRNAs involved in HCC through the comparision of miRNA expression profiling in cancerous hepatocytes with that in normal hepatocytes.37 dysregulated miRNAs were screened out by 2-fold change with a significant difference (P< 0.05). Furthermore, we demonstrated that both up-regulated miR-221 and down-regulated miR-375 were involved in hepatocarcinogenesis by modulating cell cycle, proliferation, apoptosis, invasion and clone formation. Mir-221 targets three tumor suppressors-BMF, BBC3, ANGPTL2, and functions as an oncogene, while miR-375 targets two oncogenes-LAMC1, PDGFC, and functions as a tumor suppressor. Therapeutic modulation by lentivirus-mediated-anti-miR-221 or cholesterol modified miR-375 mimics can significantly suppress the growth of hepatoma xenografts in nude mice.
Conclusions We emphasized that HCC-related miRNAs identified by comparison of the expression profiling of miRNAs in liver cancer cell lines versus normal hepatocytes could be more representative and informative in hepatocarcinogenesis. Furthermore, our findings highlight the therapeutic potential of lentivirus-mediated-anti-miR-221 and cholesterol modified miR-375 for HCC. With all these results, we believe that the field of microRNA based diagnosis and therapeutics has incredible promise in the fight against HCC.
方法应用Agilent human miRNA array分析7株肝癌细胞株和3例原代正常肝细胞的miRNA表达谱,通过比较得到肝癌中异常表达的miRNAs,并用TaqMan qRT-PCR进行确认。在细胞水平上转染miRNA前体或抑制剂以及相应的对照miRNA,通过细胞增殖、细胞周期和凋亡分析、细胞迁移与侵袭以及克隆形成实验,探讨这些异常表达的miRNAs在肝癌发生发展中的作用。利用生物信息学技术如miRBase、TargetScan、PicTar等数据库,预测miRNA的作用靶点,再通过SYBR Green定量PCR、Western Blot以及Luciferase精确靶点实验,验证肝癌相关miRNAs的作用靶点,从而阐明这些异常表达的miRNAs在肝癌发生中,类似于抑癌基因或癌基因的分子作用机制。利用肝癌裸鼠移植瘤模型,探索这些异常表达的miRNAs分子或其拮抗剂在肝癌治疗中的潜力。
结果通过比较肝癌细胞株与原代正常肝细胞的miRNA表达谱差异获得肝癌异常表达miRNAs 37个(2-fold change,P< 0.05)。我们证实上调的miR-221和下调的miR-375通过调控细胞增殖、周期、凋亡、迁移侵袭以及克隆形成参与了肝癌的发生发展。miR-221作用其靶点BMF、BBC3和ANGPTL2而作用类似于癌基因,miR-375作用其靶点LAMC1和PDGFC而作用类似于抑癌基因。治疗性的应用慢病毒介导的anti-miR-221或胆固醇修饰的miR-375类似物可以有效的抑制肝癌裸鼠移植瘤的生长。
结论通过比较肝癌细胞株与原代正常肝细胞的miRNA表达谱差异可以高效鉴定出肝癌特异性的miRNAs。这些异常表达的miRNAs通过作用其靶点,调节了肿瘤发生相关的重要生物学过程,从而参与了肝癌的发生和发展。实验结果显示慢病毒介导的anti-miR-221或胆固醇修饰的miR-375类似物对治疗肝癌有良好的潜力。所有这些,使我们相信基于miRNAs的诊断和治疗在抗肝癌研究中具有诱人的前景。
Objective Hepatocellular carcinoma (HCC) is a worldwide human malignancy with high morbidity and mortality but lack of effective targeted therapies. Current diagnosis biomarkers of HCC are maily depend on alpha-fetoportien (AFP), but 20%to 40%HCC patients'AFP was negative or low positive. Up to now, surgical resection or liver transplantation remains the first choice for the treatment of HCC; however, only about 20% of patients with HCC are eligible for surgical intervention at the time of diagnosis. Moreover, patients who underwent curative resection often have a high frequency of relapse, and postoperative 5-year survival is only 30%-40%. The outcomes of HCC patients are still so dismal due to our limited knowledge in its molecular pathogenesis, the difficulty in detecting the disease at its early stages and the lack of effective therapeutics. Therefore, studies aimed at the definition of the mechanisms associated with hepatocarcinogenesis, identification of new biomarkers for early diagnosis and development of more effective therapeutic interventions are urgently needed. MiRNAs are evolutionarily conserved, small noncoding RNAs that are believed to play fundamental roles in various biological processes through regulation of gene expression at the level of post-transcription. Alterations in miRNA expression can modulate key cellular processes involved in tumorigenesis; so systematic evaluation of changes in global miRNA expression in phenotypically defined tumors could provide insight into basic mechanisms of tumor formation and progression. In this report, we systematically studied the aberrantly expressed miRNAs involved in HCC through the comparison of miRNA expression profiling in cancerous hepatocytes with that in normal primary human hepatocytes and emphasized the functions, targets and therapeutic potential of these aberrantly expressed miRNAs.
Methods The expression profiles containing 723 human miRNAs were determined in 7 liver cancer cell lines and 3 cases of normal primary human hepatocytes (PHHC) using Agilent human miRNA array (Version 2). MiRNA expression profiles of cancerous hepatocytes were compared with that of normal hepatocytes to indentify the aberrantly expressed miRNAs. These results were further confirmed by TaqMan qRT-PCR. To assess the role of dysregulated miRNAs in hepatocarcinogenesis, hepatoma cell lines were transfected with pre-miRNAs, anti-miRNAs or their matched negative control and further tested by cell proliferation, cell cycle and apoptosis, clonogenicity, cell migration and invasion assaies. The putative miRNA targets were predicted by using algorithms miRBase, TargetScan and PicTar and verfied by SYBR Green qRT-PCR, Western Blot Analysis and Luciferase Assay. The model of hepatoma xenografts in nude mice was established to explore the therapeutic potential of antisense-mediated inhibition of oncogenic miRNAs or replacement therapy of tumor suppressor miRNAs for HCC.
Results we reported a previously unrecognized paradigm for identifying aberrantly expressed miRNAs involved in HCC through the comparision of miRNA expression profiling in cancerous hepatocytes with that in normal hepatocytes.37 dysregulated miRNAs were screened out by 2-fold change with a significant difference (P< 0.05). Furthermore, we demonstrated that both up-regulated miR-221 and down-regulated miR-375 were involved in hepatocarcinogenesis by modulating cell cycle, proliferation, apoptosis, invasion and clone formation. Mir-221 targets three tumor suppressors-BMF, BBC3, ANGPTL2, and functions as an oncogene, while miR-375 targets two oncogenes-LAMC1, PDGFC, and functions as a tumor suppressor. Therapeutic modulation by lentivirus-mediated-anti-miR-221 or cholesterol modified miR-375 mimics can significantly suppress the growth of hepatoma xenografts in nude mice.
Conclusions We emphasized that HCC-related miRNAs identified by comparison of the expression profiling of miRNAs in liver cancer cell lines versus normal hepatocytes could be more representative and informative in hepatocarcinogenesis. Furthermore, our findings highlight the therapeutic potential of lentivirus-mediated-anti-miR-221 and cholesterol modified miR-375 for HCC. With all these results, we believe that the field of microRNA based diagnosis and therapeutics has incredible promise in the fight against HCC.
引文
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